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1.
Small Methods ; 7(4): e2201452, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36808832

RESUMO

The performance of fluorescence immunostaining is physically limited by the brightness of organic dyes, whereas fluorescence labeling with multiple dyes per antibody can lead to dye self-quenching. The present work reports a methodology of antibody labeling by biotinylated zwitterionic dye-loaded polymeric nanoparticles (NPs). A rationally designed hydrophobic polymer, poly(ethyl methacrylate) bearing charged, zwitterionic and biotin groups (PEMA-ZI-biotin), enables preparation of small (14 nm) and bright fluorescent biotinylated NPs loaded with large quantities of cationic rhodamine dye with bulky hydrophobic counterion (fluorinated tetraphenylborate). The biotin exposure at the particle surface is confirmed by Förster resonance energy transfer with dye-streptavidin conjugate. Single-particle microscopy validates specific binding to biotinylated surfaces, with particle brightness 21-fold higher than quantum dot-585 (QD-585) at 550 nm excitation. The nanoimmunostaining method, which couples biotinylated antibody (cetuximab) with bright biotinylated zwitterionic NPs through streptavidin, significantly improves fluorescence imaging of target epidermal growth factor receptors (EGFR) on the cell surface compared to a dye-based labeling. Importantly, cetuximab labeled with PEMA-ZI-biotin NPs can differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal from labeled antibodies, and thus become a useful tool in the high-sensitivity detection of disease biomarkers.


Assuntos
Corantes Fluorescentes , Nanopartículas , Corantes Fluorescentes/química , Biotina/química , Biotina/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Cetuximab , Nanopartículas/química , Polímeros/química
2.
Pharmaceutics ; 14(10)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36297416

RESUMO

Nucleic-acid aptamers are of strong interest for diagnosis and therapy. Compared with antibodies, they are smaller, stable upon variations in temperature, easy to modify, and have higher tissue-penetration abilities. However, they have been little described as detection probes in histology studies of human tissue sections. In this study, we performed fluorescence imaging with two aptamers targeting cell-surface receptors EGFR and integrin α5ß1, both involved in the aggressiveness of glioblastoma. The aptamers' cell-binding specificities were confirmed using confocal imaging. The affinities of aptamers for glioblastoma cells expressing these receptors were in the 100-300 nM range. The two aptamers were then used to detect EGFR and integrin α5ß1 in human glioblastoma tissues and compared with antibody labeling. Our aptafluorescence assays proved to be able to very easily reveal, in a one-step process, not only inter-tumoral glioblastoma heterogeneity (differences observed at the population level) but also intra-tumoral heterogeneity (differences among cells within individual tumors) when aptamers with different specificities were used simultaneously in multiplexing labeling experiments. The discussion also addresses the strengths and limitations of nucleic-acid aptamers for biomarker detection in histology.

3.
Cells ; 10(11)2021 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-34831480

RESUMO

EGFR (epidermal growth factor receptor), a member of the ErbB tyrosine kinase receptor family, is a clinical therapeutic target in numerous solid tumours. EGFR overexpression in glioblastoma (GBM) drives cell invasion and tumour progression. However, clinical trials were disappointing, and a molecular basis to explain these poor results is still missing. EGFR endocytosis and membrane trafficking, which tightly regulate EGFR oncosignaling, are often dysregulated in glioma. In a previous work, we showed that EGFR tyrosine kinase inhibitors, such as gefitinib, lead to enhanced EGFR endocytosis into fused early endosomes. Here, using pharmacological inhibitors, siRNA-mediated silencing, or expression of mutant proteins, we showed that dynamin 2 (DNM2), the small GTPase Rab5 and the endocytosis receptor LDL receptor-related protein 1 (LRP-1), contribute significantly to gefitinib-mediated EGFR endocytosis in glioma cells. Importantly, we showed that inhibition of DNM2 or LRP-1 also decreased glioma cell responsiveness to gefitinib during cell evasion from tumour spheroids. By highlighting the contribution of endocytosis proteins in the activity of gefitinib on glioma cells, this study suggests that endocytosis and membrane trafficking might be an attractive therapeutic target to improve GBM treatment.


Assuntos
Endocitose , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Linhagem Celular Tumoral , Dinamina II/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Inativação Gênica , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
4.
Cancers (Basel) ; 13(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919725

RESUMO

The nonreceptor tyrosine kinase FAK is a promising target for solid tumor treatment because it promotes invasion, tumor progression, and drug resistance when overexpressed. Investigating the role of FAK in human melanoma cells, we found that both in situ and metastatic melanoma cells strongly express FAK, where it controls tumor cells' invasiveness by regulating focal adhesion-mediated cell motility. Inhibiting FAK in human metastatic melanoma cells with either siRNA or a small inhibitor targeting the kinase domain impaired migration but led to increased invadopodia formation and extracellular matrix degradation. Using FAK mutated at Y397, we found that this unexpected increase in invadopodia activity is due to the lack of phosphorylation at this residue. To preserve FAK-Src interaction while inhibiting pro-migratory functions of FAK, we found that altering FAK-paxillin interaction, with either FAK mutation in the focal adhesion targeting (FAT) domain or a competitive inhibitor peptide mimicking paxillin LD domains drastically reduces cell migration and matrix degradation by preserving FAK activity in the cytoplasm. In conclusion, our data show that targeting FAK-paxillin interactions could be a potential therapeutic strategy to prevent metastasis formation, and molecules targeting this interface could be alternative to inhibitors of FAK kinase activity which display unexpected effects.

5.
Biophys Rep (N Y) ; 1(2): 100021, 2021 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36425460

RESUMO

Over the last decades, several techniques have been developed to study cell adhesion; however, they present significant shortcomings. Such techniques mostly focus on strong adhesion related to specific protein-protein associations, such as ligand-receptor binding in focal adhesions. Therefore, weak adhesion, related to less specific or nonspecific cell-substrate interactions, are rarely addressed. Hence, we propose in this work a complete investigation of cell adhesion, from highly specific to nonspecific adhesiveness, using variable-angle total internal reflection fluorescence (vaTIRF) nanoscopy. This technique allows us to map in real time cell topography with a nanometric axial resolution, along with cell cortex refractive index. These two key parameters allow us to distinguish high and low adhesive cell-substrate contacts. Furthermore, vaTIRF provides cell-substrate binding energy, thus revealing a correlation between cell contractility and cell-substrate binding energy. Here, we highlight the quantitative measurements achieved by vaTIRF on U87MG glioma cells expressing different amounts of α 5 integrins and distinct motility on fibronectin. Regarding integrin expression level, data extracted from vaTIRF measurements, such as the number and size of high adhesive contacts per cell, corroborate the adhesiveness of U87MG cells as intended. Interestingly enough, we found that cells overexpressing α 5 integrins present a higher contractility and lower adhesion energy.

6.
Cell Mol Life Sci ; 78(6): 2949-2962, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33151388

RESUMO

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.


Assuntos
Endocitose/efeitos dos fármacos , Gefitinibe/farmacologia , Integrina alfa5beta1/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/genética , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo
7.
Mol Ther Nucleic Acids ; 17: 63-77, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31226519

RESUMO

Nucleic acid aptamers are often referred to as chemical antibodies. Because they possess several advantages, like their smaller size, temperature stability, ease of chemical modification, lack of immunogenicity and toxicity, and lower cost of production, aptamers are promising tools for clinical applications. Aptamers against cell surface protein biomarkers are of particular interest for cancer diagnosis and targeted therapy. In this study, we identified and characterized RNA aptamers targeting cells expressing integrin α5ß1. This αß heterodimeric cell surface receptor is implicated in tumor angiogenesis and solid tumor aggressiveness. In glioblastoma, integrin α5ß1 expression is associated with an aggressive phenotype and a decrease in patient survival. We used a complex and original hybrid SELEX (selective evolution of ligands by exponential enrichment) strategy combining protein-SELEX cycles on the recombinant α5ß1 protein, surrounded by cell-SELEX cycles using two different cell lines. We identified aptamer H02, able to differentiate, in cyto- and histofluorescence assays, glioblastoma cell lines, and tissues from patient-derived tumor xenografts according to their α5 expression levels. Aptamer H02 is therefore an interesting tool for glioblastoma tumor characterization.

8.
Cancers (Basel) ; 11(5)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31109009

RESUMO

Integrins contribute to cancer progression and aggressiveness by activating intracellular signal transduction pathways and transducing mechanical tension forces. Remarkably, these adhesion receptors share common signaling networks with receptor tyrosine kinases (RTKs) and support their oncogenic activity, thereby promoting cancer cell proliferation, survival and invasion. During the last decade, preclinical studies have revealed that integrins play an important role in resistance to therapies targeting RTKs and their downstream pathways. A remarkable feature of integrins is their wide-ranging interconnection with RTKs, which helps cancer cells to adapt and better survive therapeutic treatments. In this context, we should consider not only the integrins expressed in cancer cells but also those expressed in stromal cells, since these can mechanically increase the rigidity of the tumor microenvironment and confer resistance to treatment. This review presents some of these mechanisms and outlines new treatment options for improving the efficacy of therapies targeting RTK signaling.

9.
Nanotechnology ; 30(18): 184005, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-30650397

RESUMO

Therapeutic monoclonal antibodies benefit to patients and the conjugation to gold nanoparticles (AuNPs) might bring additional activities to these macromolecules. However, the behavior of the conjugate will largely depend on the bulkiness of the AuNP and small sizes are moreover preferable for diffusion. Water-soluble thiolate-protected AuNPs having diameters of 2-3 nm can be synthesized with narrow polydispersity and can selectively react with incoming organic thiols via a SN2-like mechanism. We therefore synthesized a mixed thionitrobenzoic acid- , thioaminobenzoic acid-monolayered AuNP of 2.4 nm in diameter and developed a site-selective conjugation strategy to link the AuNP to Cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody used in clinic. The water-soluble 80 kDa AuNP was fully characterized and then reacted to the hinge area of Cetuximab, which was selectively reduced using mild concentration of TCEP. The conjugation proceeded smoothly and could be analyzed by polyacrylamide gel electrophoresis, indicating the formation of a 1:1 AuNP-IgG conjugate as the main product. When added to EGFR expressing glioblastoma cells, the AuNP-Cetuximab conjugate selectively bound to the cell surface receptor, inhibited EGFR autophosphorylation and entered into endosomes like Cetuximab. Altogether, we describe a simple and robust protocol for a site-directed conjugation of a thiolate-protected AuNP to Cetuximab, which could be easily monitored, thereby allowing to assess the quality of the product formation. The conjugated 2.4 nm AuNP did not majorly affect the biological behavior of Cetuximab, but provided it with the electronic properties of the AuNP. This offers the ability to detect the tagged antibody and opens application for targeted cancer radiotherapy.


Assuntos
Cetuximab , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Ouro , Nanopartículas Metálicas , Linhagem Celular Tumoral , Cetuximab/química , Cetuximab/farmacologia , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Ouro/química , Ouro/farmacologia , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Proteínas de Neoplasias/metabolismo , Tamanho da Partícula
10.
Bioconjug Chem ; 30(1): 192-199, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30562000

RESUMO

Staining of the plasma membrane (PM) is essential in bioimaging, as it delimits the cell surface and provides various information regarding the cell morphology and status. Herein, the lipophilicity of a green emitting BODIPY fluorophore was tuned by gradual functionalization with anchors composed of zwitterionic and aliphatic groups, thus yielding three different amphiphilic dyes. We found that BODIPY bearing one or three anchors failed in efficiently staining the PM: the derivative with one anchor showed low affinity to PM and exhibited strong fluorescence in water due to high solubility, whereas BODIPY with three anchors aggregated strongly in media and precipitated before binding to the PM. In sharp contrast, the BODIPY bearing two anchors (B-2AZ, MemBright-488) formed virtually nonfluorescent soluble aggregates in aqueous medium that quickly deaggregated in the presence of PM, leading to a bright soluble molecular form (quantum yield of 0.92). This fluorogenic response allowed for efficient probing of the PM at low concentration (20 nM) with high signal to background ratio images in mono- as well as two-photon excitation microscopy. B-2AZ proved to selectively stain the PM in a more homogeneous manner than the commercially available fluorescently labeled lectin WGA. Finally, it was successfully used in 3D-imaging to reveal fine intercellular tunneling nanotubes in KB cells and to stain the PM in glioblastoma cells in spheroids.


Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Membrana Celular/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência
11.
Oncotarget ; 9(73): 33788-33803, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30333910

RESUMO

Despite post-operative radio-chemotherapy, glioblastoma systematically locally recurs. Tumors contacting the periventricular zone (PVZ) show earlier and more distant relapses than tumors not contacting the PVZ. Since glioblastoma stem-like cells (GSCs) have been proposed to play a major role in glioblastoma recurrence, we decided to test whether GSC migration properties could be different according to their anatomical location (PVZ+/PVZ-). For that purpose, we established paired cultures of GSCs from the cortical area (CT) and the PVZ of glioblastoma patient tumors. We demonstrated that PVZ GSCs possess higher migration and invasion capacities than CT GSCs. We highlighted specific transcriptomic profiles in PVZ versus CT populations and identified a down-regulation of the RhoGTPase, RND1 in PVZ GSCs compared to CT GSCs. Overexpression of RND1, dramatically inhibited PVZ GSC migration and conversely, downregulation of RND1 increased CT GSC migration. Additionally, transcriptomic analyses also revealed a down-regulation of RND1 in glioblastoma compared to normal brain. Using the glioblastoma TCGA database, low levels of RND1 were also shown to correlate with a decreased overall survival of patients. Finally, based on signaling pathways activated in patients with low levels of RND1, we identified an RND1 low signature of six genes (MET, LAMC1, ITGA5, COL5A1, COL3A1, COL1A2) that is an independent prognostic factor in glioblastoma. These findings contribute to explain the shorter time to progression of patients with PVZ involvement and, point out genes that establish the RND1 low signature as key targets genes to impede tumor relapse after treatment.

12.
Oncotarget ; 7(38): 62194-62207, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27613837

RESUMO

The Wnt/beta catenin pathway has been highlighted as an important player of brain tumors aggressiveness and resistance to therapies. Increasing knowledges of the regulation of beta-catenin transactivation point out its hub position in different pathophysiological outcomes in glioma such as survival and migration. Crosstalks between integrins and beta-catenin pathways have been suggested in several tumor tissues. As we demonstrated earlier that α5ß1 integrin may be considered as a therapeutic target in high grade glioma through its contribution to glioma cell migration and resistance to chemotherapy, we addressed here the potential relationship between α5ß1 integrin and beta-catenin activation in glioma cells. We demonstrated that overexpression and activation by fibronectin of α5ß1 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that α5ß1 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of α5ß1 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that α5ß1 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma.


Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular , Glioma/patologia , Integrina alfa5beta1/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Apoptose , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibronectinas/metabolismo , Glioma/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Imuno-Histoquímica , Integrina alfa5beta1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tanquirases/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , beta Catenina/antagonistas & inibidores
13.
Cancer Lett ; 376(2): 328-38, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27063097

RESUMO

Glioblastoma multiform (GBM) is the most common and most aggressive primary brain tumor. The fibronectin receptor, α5 integrin is a pertinent novel therapeutic target. Despite numerous data showing that α5 integrin support tumor cell migration and invasion, it has been reported that α5 integrin can also limit cell dispersion by increasing cell-cell interaction. In this study, we showed that α5 integrin was involved in cell-cell interaction and gliomasphere formation. α5-mediated cell-cell cohesion limited cell dispersion from spheroids in fibronectin-poor microenvironment. However, in fibronectin-rich microenvironment, α5 integrin promoted cell dispersion. Ligand-occupied α5 integrin and fibronectin were distributed in fibril-like pattern at cell-cell junction of evading cells, forming cell-cell fibrillar adhesions. Activated focal adhesion kinase was not present in these adhesions but was progressively relocalized with α5 integrin as cell migrates away from the spheroids. α5 integrin function in GBM appears to be more complex than previously suspected. As GBM overexpressed fibronectin, it is most likely that in vivo, α5-mediated dissemination from the tumor mass overrides α5-mediated tumor cell cohesion. In this respect, α5-integrin antagonists may be useful to limit GBM invasion in brain parenchyma.


Assuntos
Neoplasias Encefálicas/metabolismo , Adesão Celular , Comunicação Celular , Movimento Celular , Junções Célula-Matriz/metabolismo , Matriz Extracelular/metabolismo , Integrina alfaV/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrina alfaV/genética , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais , Esferoides Celulares , Fatores de Tempo , Transfecção
14.
Front Pharmacol ; 6: 279, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26635609

RESUMO

Integrins belong to a large family of αß heterodimeric transmembrane proteins first recognized as adhesion molecules that bind to dedicated elements of the extracellular matrix and also to other surrounding cells. As important sensors of the cell microenvironment, they regulate numerous signaling pathways in response to structural variations of the extracellular matrix. Biochemical and biomechanical cues provided by this matrix and transmitted to cells via integrins are critically modified in tumoral settings. Integrins repertoire are subjected to expression level modifications, in tumor cells, and in surrounding cancer-associated cells, implicated in tumor initiation and progression as well. As critical players in numerous cancer hallmarks, defined by Hanahan and Weinberg (2011), integrins represent pertinent therapeutic targets. We will briefly summarize here our current knowledge about integrin implications in those different hallmarks focusing primarily on ß1 integrins.

15.
Oncotarget ; 6(39): 41884-901, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26474461

RESUMO

Distant metastases arise in 20-30% of patients with squamous cell carcinoma of the head and neck (HNSCC) in the 2 years following treatment. Therapeutic options are limited and the outcome of the patients is poor. The identification of predictive biomarkers of patient at risk for distant metastasis and therapies are urgently needed. We previously identified a clinical subgroup, called "R1" characterized by high propensity for rapid distant metastasis. Here, we showed that "R1" patients do not or at very low level express caveolin-1 (Cav1). Low or no expression of Cav1 is of bad prognosis. Disappearance of Cav1 enables cells to undergo epithelial-mesenchymal transition (EMT). EMT is associated with enhanced migration and invasion. Our study uncovered a new target, α5ß1 integrin. Targeting α5ß1 integrins might not only prevent metastasis of HNSCC but also delay the development of the primary tumor by reducing tumor cell viability. Cav1 detection might be taken into consideration in the future in the clinic not only to identify patients at high risk of metastasis but also to select patient who might benefit from an anti-integrin therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Caveolina 1/metabolismo , Movimento Celular , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço/metabolismo , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Carcinoma de Células Escamosas/terapia , Caveolina 1/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Integrina alfa5beta1/metabolismo , Estimativa de Kaplan-Meier , Prognóstico , Interferência de RNA , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fatores de Tempo , Transfecção
16.
Biochim Biophys Acta ; 1840(9): 2978-87, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24810979

RESUMO

BACKGROUND: Integrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5ß1 and αvß3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5ß1 and αvß3/ß5 purified integrins. METHODS: Small antagonists were either selective for α5ß1 integrin, for αvß3/ß5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5ß1 integrin in the biological assays. RESULTS: U87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5ß1 or αvß3/ß5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5ß1 integrin and was inhibited selectively by α5ß1 integrin antagonists but increased by αvß3/ß5 integrin antagonists. CONCLUSIONS: We provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions. GENERAL SIGNIFICANCE: Our data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5ß1 integrin-dependent cell migration.


Assuntos
Antineoplásicos , Glioma/tratamento farmacológico , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Cadeias beta de Integrinas , Proteínas de Neoplasias/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Integrina alfa5beta1/biossíntese , Integrina alfa5beta1/genética , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética
17.
Cancer Lett ; 336(2): 307-18, 2013 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-23523610

RESUMO

Integrins emerge nowadays as crucial actors of tumor aggressiveness and resistance to therapies. Integrin α5ß1, the fibronectin receptor, determines malignant properties of colon carcinoma which is one of the most important causes of cancer-related deaths in the world. Here we show that inhibition of α5 integrin subunit expression by siRNA or α5ß1 integrin function by specific antagonist affects the survival of HCT116 colon cancer cells. We also evidence that pharmacological reactivation of the tumor suppressor p53 by Nutlin-3a inhibits specifically the expression of the α5 integrin subunit both at the transcriptional and protein level. Inversely repression of α5 integrin modulates p53 activity. A clear relationship between p53 activation by Nutlin-3a, α5 repression and cell survival is shown. No such effects are obtained in cells lacking p53 or when another non-genotoxic activator of p53, RITA, is used. Our results emphasize the crucial role of α5ß1 integrin in colon tumors. Data also suggest that interfering with the integrin α5ß1 through the reactivation of p53 by Nutlin-3a may be of valuable interest as a new therapeutic option for colon tumors expressing high level of the integrin and a wild type p53.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Imidazóis/farmacologia , Integrina alfa5/biossíntese , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células HCT116 , Humanos , Integrina alfa5/genética , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , Transfecção
18.
J Cell Sci ; 125(Pt 18): 4264-77, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22718344

RESUMO

α6ß4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6ß4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of α6ß4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6ß4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Hemidesmossomos/metabolismo , Queratinócitos/citologia , Queratinócitos/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores Purinérgicos P2Y2/metabolismo , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hemidesmossomos/efeitos dos fármacos , Humanos , Integrina beta4/metabolismo , Queratinócitos/efeitos dos fármacos , Modelos Biológicos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Uridina Trifosfato/farmacologia , Quinases raf/metabolismo
19.
Mol Biol Cell ; 21(6): 946-55, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20089844

RESUMO

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.


Assuntos
Movimento Celular/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Queratinócitos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Cortactina/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Queratinócitos/citologia , Peptídeos Cíclicos/metabolismo , Fosfolipase C beta/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y2 , Uridina Trifosfato/metabolismo
20.
Cancer Lett ; 285(2): 174-81, 2009 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-19501458

RESUMO

Cell adhesion molecules, including cadherins and integrins, play an essential role during tumor progression and represent potential targets for the development of new therapeutic agents. We previously showed that lebectin, a C-type lectin protein (CLP) issued from Macrovipera lebectina snake venom, inhibits integrin-mediated migration of IGR39 melanoma cells. Here we assessed whether lebectin modulates cell-cell adhesion. We demonstrated that lebectin promotes N-cadherin/catenin complex reorganization at cell-cell contacts, inducing a strengthening of intercellular adhesion. This reorganization is associated to phosphorylation of beta-catenin on tyrosine 142 residue. Interestingly, lebectin acts on N-cadherin-mediated cell-cell contacts through PI3K/Akt pathway. This effect could contribute to the blockage of tumor cell migration previously observed.


Assuntos
Caderinas/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Venenos de Víboras/farmacologia , Western Blotting , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Imunoprecipitação , Lectinas Tipo C , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia
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